JPH06104073B2 - Manufacturing method of test piece for body fluid component inspection - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Object of the Invention (Industrial field of application) The present invention provides a test piece for testing body fluid components, which can be widely used in the fields of pharmaceutical chemistry and analytical chemistry, such as test agents and diagnostic agents. It relates to a manufacturing method.

(Prior art) In the fields of pharmaceutical chemistry and analytical chemistry such as test drugs and diagnostic agents, pathological components in body fluids are measured under relatively mild conditions without using strong reagents such as strong acids or strong alkalis. Therefore, the assay method using biologically active protein such as enzyme, hormone, substance capable of participating in antigen-antibody reaction or hapten antibody reaction, coagulation factor, etc.
It is widely used, including the glucose detection products described in Japanese Examined Patent Publication No. 45-1878.

These test pieces are usually obtained by impregnating an absorbent material such as filter paper with the test agent composition and drying it, and then cutting it into small pieces and adhering the small pieces to a substrate with high mechanical strength such as a plastic film. It is manufactured by sticking it using an agent or the like, or by printing the test drug composition on the base material. However, the above method has a drawback that the manufacturing process is extremely complicated and the cost is high.

As the above-mentioned method, an enzyme which is a biologically active protein is used and a test agent is printed on a substrate as an aqueous composition (see Japanese Patent Publication No. 44-25953), or the test agent is water-alcoholic. There is a method of printing on a substrate as a composition (see Japanese Utility Model Publication No. 57-79767, etc.). However, since the test agent composition contains a large amount of water in both cases, after printing, it can be stored at room temperature or at a low temperature lower than room temperature for a long time. It had to be dried for a long time, which was inferior in workability and productivity, and there was a drawback that the enzyme was inactivated by heating and drying in an attempt to dry in a short time. In order to dry at a lower temperature in a short time, the enzyme
A method of printing a composition dispersed in a non-aqueous solvent such as butanol on a substrate as a detection agent (JP-A-58-209995, JP-A-60-178358, JP-A-61-284661) (See, etc.) is also proposed, but even though it is low temperature and short time,
Since the enzyme having a low thermostability is heated, most of the enzyme in the test drug composition is inactivated and the amount of effective enzyme is reduced, so that there is a drawback that a large amount of expensive enzyme must be used.

(Problems to be Solved by the Invention) Since the present invention can be cured and dried at low temperature in a short time, biologically active proteins such as enzymes in test drug compositions are not inactivated, and expensive biological activity is not caused. It is intended to provide a method for producing a test piece for inspecting body fluid components, which requires only a required amount of protein and has good workability and productivity.

[Structure of Invention]

(Means for Solving the Problem) The present invention prints a printing ink composition comprising a biologically active protein, an indicator and a radiation-curable compound on a substrate, and prints the printed printing ink composition with active radiation. A method for producing a test piece for body fluid component inspection, which is characterized by being cured by irradiation.

In the present invention, as the substrate, paper such as filter paper, art paper, synthetic paper, non-woven fabric, woven cloth, plastic film, foamed plastic sheet, metal foil, or a laminated body of these is used.

In the present invention, as the biologically active protein, a plant or animal active protein, or a modified form thereof in which a chemical substance such as a crosslinking agent is grafted is used. Such biologically active proteins include:
There are enzymes, hormones, antigen-antibody reactions or biological proteins that cause hapten antibody reactions, coagulation factors, etc.,
Enzymes include pathological components contained in body fluids such as blood in the body, or in urine, such as inorganic components such as calcium and magnesium, glucose such as glucose, cholesterol, urea, uric acid, creatinine, and neutral. By reacting with fats, phospholipids, ammonia, lactic acid, pyruvic acid, sialic acid, GPT, GOT, cholinesterase, leucine aminopeptidase, amylase, LCAT, creatine phosphokinase, etc., which changes the color tone of the indicator, or by the above reaction The produced substance reacts with other enzymes to change the color tone of the indicator, or as a result of these reactions, it emits light or changes in spectrum, and is detected by a spectrophotometer such as visible ultraviolet absorption spectrum or fluorescence spectrum. What can be analyzed is used. The biologically active protein that causes an antigen-antibody reaction or a hapten antibody reaction is not particularly limited, and is collectively referred to as hapten or immunoglobulin, which are substances that show an immune response when bound to a high molecular substance such as protein. Such as antibodies, humans, monkeys, mice, rats,
Immunoglobulins obtained from dogs, rabbits, cows, horses, birds, reptiles, amphibians, fishes, ornithopods can be used, but are not limited thereto.

In the present invention, as the indicator, a compound (one-component indicator) that emits light, develops a color, shows a color change, or undergoes a spectrum change by an enzymatic reduction reaction, or a compound produced by a redox reaction is further included. When combined with the compound (coupler), it emits light, develops color,
It is a compound (developing agent) that exhibits a color change or a spectrum change. One-component indicators include o-toluidine, 3,3 ', 5,5'-tetramethylbenzine, guaiac butter, p-anisidine, N, N-dimethyl p-phenylenediamine, 2,7-diaminofluorene, 2 2,2'-dianobis (3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dichlorophenol, α-naphthol, potassium iodide and the like. Examples of developers include 4-aminoantipyrine, 3-methyl-2-benzothiazolinone hydrazone, tetramethylbenzidine, and N, N-dimethyl-p-phenylenediamine. As couplers, 1,7-dihydroxynaphthalene, 1,8-dihydroxy-3,6-disulfonic acid naphthalene, 8-amino-1,1-naphthol-3,6
-Naphthols such as phthalene disulfonate, dimeraniline, diethylaniline, N-methyl-N-hydroxyethylaniline, N-methyl-N-hydroxyethyl-
Anilines such as m-toluidine, N, N-dimethyl-m-anidin, phenols, p-chlorophenol, 2,6-dichlorophenol, guaiacol, pyrogallol, phenols such as o-phenylphenol, 1-naphthol-3 ,
There are sulfonic acids such as 6-disulfonic acid, 1-aminonaphthalene-5-sulfonic acid and 1-aminonaphthalene-6-sulfonic acid, and salts of these sulfonic acids with higher alkylamines.

In the present invention, the radiation-curable compound includes (meth)
Acrylic acid, (meth) acrylic acid salts, (meth) acrylic acid alkyl esters, (meth) acrylic acid glycidyl ester, (meth) acrylic acid hydroxyalkyl esters, (meth) acrylonitrile, (meth) acrylamide, styrene, α -Alkyl styrenes, vinyl acetate, vinylidene chloride, vinyl ethers, allyl alcohol, etc., or substitution products thereof, poly (meth) acrylates such as di (meth) acrylates, tri (meth) acrylates, di (meth) There are acrylamides, diallyl (iso) phthalate, diallyl (iso) phthalate oligomers, divinylbenzene, triallyl compounds, etc., or a mixture thereof.

In the present invention, a printing ink composition as a test and inspection agent is required within the range that does not impair the performance of the biological fluid component test piece of the present invention, in addition to the above biologically active protein, indicator, and radiation curable compound. Depending on the type, silica, inorganic fillers such as kaolin, pH buffers, surfactants, antioxidants, carboxymethyl cellulose for improving liquid absorption and liquid retention properties for improving liquid absorption and liquid retention properties Water-soluble polymers such as sodium salt, methylcellulos, agar, sodium alginate, sodium polyacrylate, gelatin, starch, dyes and pigments, polymerization inhibitors, photopolymerization initiators, photopolymerization accelerators, lubricants, thermoplastic resins, It can be obtained by adding a small amount of water, an organic solvent, a small amount of an additive such as a thermosetting resin, and thoroughly mixing or kneading. The amount of indicator in the printing ink composition thus obtained is 0.
It is preferably from 1 to 5% by weight.

The printing ink composition as a test test agent thus obtained may be used alone or in appropriate combination on the substrate, or two or more of them may be used alone or in combination on different portions of the substrate. Printing, lithographic printing, gravure printing,
Printing is performed by an appropriate printing method selected according to the type of substrate and printing ink composition such as flexographic printing and screen printing, and the printed portion of the substrate is irradiated with actinic radiation such as ultraviolet rays, electron beams, and X-rays. Then, this is cured, and if necessary, cut into an appropriate size to obtain the test piece for body fluid component inspection of the present invention. When the actinic radiation is ultraviolet rays, it is preferable from the viewpoint of curability that the printing ink composition contains a photopolymerization initiator and, if necessary, a photopolymerization accelerator. When the substrate has actinic radiation transparency, the actinic radiation is applied from the side opposite to the printed surface of the substrate,
The printed portion can also be cured.

By using the thus obtained test piece for testing body fluid components, pathological components contained in body fluid such as blood or urine in vivo, for example, inorganic components such as calcium and magnesium, glucose Glucose, cholesterol, urea, uric acid, creatinine, neutral fat, phospholipids, ammonia, lactic acid, pyruvate, sialic acid, GPT, GOT, cholinesterase, leucine aminopeptidase, amylase, LCAT, creatine phosphokinase, etc. can do.

(Examples) Hereinafter, the present invention will be described with reference to Examples. In the examples, “part” means “part by weight”.

Example 1. Preparation of buffer dispersion A Vinylpyrrolidone 125 parts Citric acid monohydrate 5 parts Trisodium citrate dihydrate 20 parts 2,6-Di-tert-butylhydroxytoluene 0.07 parts Mixture of the above composition Was dispersed with a ball mill for 2 days to obtain a milky white buffer dispersion liquid A.

Preparation of Dispersion B Buffer Dispersion A 15 parts Vinyl alcohol-acrylate block copolymer (super absorbent gel) "Sumikagel SP520" (Sumitomo Chemical Co., Ltd., trade name) 8 parts Polyethylene glycol 6000 1 part Polyvinylpyrrolidone 7 parts Glycerin diglycidyl ether diacrylate 1 part Guayak fat 0.8 parts The mixture having the above composition was mixed and dispersed using a homogenizer to obtain a dispersion B.

Preparation of Enzyme Dispersion C Phosphate buffer (pH 7.0,1 / 15M) 1 part Glucose oxidase (Type II, Sigma) 0.2 parts Peroxidase (type II, Sigma) 0.03 parts Polyethylene glycol 400 1 part The above glucose oxidase, peroxidase and polyethylene glycol were dissolved in this order in the above phosphate buffer on an ice bath to obtain a slightly turbid brownish viscous enzyme dispersion C.

Preparation of printing ink composition Add 2.23 parts of Enzyme Dispersion C to 32.8 parts of Dispersion B while ice-cooling, stir and disperse for 10 minutes with a homogenizer, then
0.5 parts of hydroxy-2-methylpropiophenone was added and stirred for 5 minutes to obtain a printing ink composition D.

Preparation and evaluation of test pieces for body fluid component inspection Using a 100-mesh polytetrafluoroethylene screen, printing ink composition D was coated on one side of a 200 μm thick transparent polystyrene film with a 1 cm width and a printing film thickness of 35 μm.
Then, screen printing was performed to obtain a printed matter. The obtained printed matter was passed through an 80 W / cm ultraviolet lamp at a speed of 6.5 m / min four times to radiate ultraviolet rays from the print side of the film to cure the printed portion. The total irradiation dose of ultraviolet rays at this time was 200 mJ / cm ² . In addition, there was no tack in the print-cured part, and the adhesion of the print-cured part to the polystyrene film substrate was good.

The obtained print-cured film was cut so that the size of the test drug area was a square of 1 cm in width and 1 cm in width, and a test piece was obtained.
The obtained test pieces were applied to glucose aqueous solutions of various concentrations.
After immersing for 2 seconds and then taking it out, the color developed after 1 minute was measured using a cyan-side light filter for reflection densitometer "B-318G".
(X-Rite, Inc., trade name). The measurement results are shown in Table 1. The numerical value of the reflection density indicates that the larger the value, the darker the color. Also, the concentration of glucose is 0
In the range of up to 500 mg / dl, a large change in reflection density was observed, and the glucose concentration can be clearly measured by visual observation as compared with the standard color.

In Table 1, the unit of glucose concentration is mg / dl. The same applies to the table below.

Also, measure the visible absorption spectrum of the obtained test piece,
Absorbances Eλ ₁ , Eλ ₂ , and Eλ ₃ at wavelengths λ ₁ = 480 nm, λ ₂ = 610 nm and λ = 740 nm, respectively, are obtained, and the actual absorbance Eλ ₂ , s at λ ₂ is subtracted from the absorbance corresponding to the baseline. Was calculated based on the equation Eλ ₂ , s = Eλ ₂ − (Eλ ₁ + Eλ ₂ ) / 2. Table 2 shows the calculation results.

From Table 2, it is understood that there is no linear relationship between the glucose concentration and Eλ ₂ , s, but the concentration of the body fluid component can be determined using the absorbance of the visible absorption spectrum.

Example 2.Butylated hydroxyanisole was used in place of 2,6-di-tert-butylhydroxytoluene, and 2-hydroxy-
A printing ink composition E was obtained in the same manner as in Example 1 except that 2-methylpropiophenone was not used. The obtained printing ink composition E was printed on a transparent polystyrene film substrate in the same manner as in Example 1, and a curtain type electron beam irradiation device (Energy Science Co., Ltd.) was used in a fluorine atmosphere at 160 kv, The printed area was cured by irradiating it with an electron beam at a dose of 7 Mrad at 5 mA. At this time, there was no tack in the print-cured part, and the adhesion of the print-cured part to the polystyrene film substrate was good.

A test piece was obtained from the obtained print-cured film in the same manner as in Example 1, and the reflection density at various glucose concentrations was measured using the obtained test piece. Table 3 shows the measured results.

In addition, the difference in color development depending on the glucose concentration was clearly distinguishable by visual observation, and it was possible to quantify glucose.

Example 3. Carried out except that butylated hydroxyanisole was used instead of 2,6-di-tert-butylhydroxytoluene, and 0.4 parts of 4-aminoantipyrine and 0.4 parts of salicylanilide were used instead of 0.8 part of guaiac butter, respectively. A printing ink composition F was obtained in the same manner as in Example 1. In the same manner as in Example 1, a transparent polystyrene film was printed, cured by irradiation with ultraviolet rays, a test piece was obtained, and a reflection density at various glucose concentrations was measured using a cyan light filter. Table 4 shows the measurement results.

Normally, the magenta (red) color can be visually distinguished sufficiently even if the difference in reflection density is not so large, and in Example 3 as well, the difference in glucose concentration could be sufficiently judged visually.

Example 4. A printing ink composition G was obtained in the same manner as in Example 3 except that 2-hydroxy-2-methylpropiophenone was not used. The resulting printing ink composition G was used in Example 2
In the same manner as described in (1), a substrate was printed, cured by electron beam irradiation, and a test piece was obtained. Table 5 shows the measured results.

From the comparison between Table 4 and Table 5, it can be seen that in these cases, the difference in color development is clearer in the test piece obtained by electron beam curing than that obtained by ultraviolet curing. It is considered that this is because the degree of crosslinking of the obtained cured product differs between electron beam curing and ultraviolet curing.

It was also found that when the irradiation dose of electron beam was changed from 7 Mrad to 10 Mrad and curing was performed, color development was suppressed.

Comparative Example 1. o-Toluidine 2.0 parts Isobutylene-maleic anhydride butanol ester "isoban # 10" (Kuraray Isopurine Chemical Co., Ltd., trade name) 2.5 parts D, L-α-tocopherol 0.1 part poly Oxyethylene sorbitan monooleate 1.2 parts Microcrystalline cellulose "Avicel SF" (Asahi Kasei Kogyo KK, trade name) 30.0 parts n-Butanol 47.0 parts Citric acid anhydride 3.2 parts preliminarily finely ground in a mortar sodium citrate anhydrous 12.0 After thoroughly finely dispersing the above mixture with a homomixer, 0.5 parts of glucose oxidase (Grade II, Toyobo Co., Ltd.) and peroxidase (Grade III,
0.1 part of Toyobo Co., Ltd. was added and finely dispersed with a homomixer to obtain a printing ink composition H. The obtained printing ink composition H was dried in the same manner as in Example 1 to give a dry film thickness of 12
Print it on a transparent polystyrene film so that it becomes 0 μm,
A test piece was obtained by heating and drying at 65 ° C. for 30 minutes in the same manner as in Example 1, and a color development test was carried out when the obtained test piece was immersed in glucose solutions of various concentrations. The average reflection density measured using a reflection densitometer was almost the same as that in Example 1, but in the region where the glucose concentration exceeded 50 mg / dl, the spotted dark blue-green portion and the light-colored portion were formed. It was observed that some areas appeared and this tendency became stronger as the glucose concentration became higher, making visual judgment difficult.

Comparative Example 2. Glucose oxidase (Grade II, Toyobo Co., Ltd.
0.5 parts and peroxidase (Grade III, manufactured by Toyobo Co., Ltd.) 0.1 part, glucose oxidase (type II, manufactured by Sigma) 0.2 parts and peroxidase (type II, manufactured by Sigma) 0.03 parts In the same manner as in Comparative Example 1 except that the printing ink composition table I was obtained, printing, heating and drying were carried out, a test piece was obtained, and a color development test was conducted using the obtained test piece. It was difficult to visually determine the glucose concentration.

〔The invention's effect〕

According to the present invention, since curing and drying can be carried out at low temperature in a short time, biologically active proteins such as enzymes in the test drug composition are not inactivated, and only a required amount of expensive biologically active protein is used. It has become possible to easily manufacture test pieces for body fluid component inspection with good workability and productivity. Further, the test piece for inspecting a body fluid component manufactured according to the present invention can be accurately inspected not only by a device but also visually.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeo Masaami 2-33 Kyobashi, Chuo-ku, Tokyo Toyo Inki Manufacturing Co., Ltd. Inspector Akira Ito

Claims (1)

[Claims] 1. A printing ink composition comprising a biologically active protein, an indicator and a radiation-curable compound is printed on a substrate, and the printed printing ink composition is cured by irradiation with actinic radiation. A method for producing a test piece for body fluid component inspection.